Cardiac excitation-contraction (EC) coupling relies on transient release of Ca from the sarcoplasmic reticulum (SR) causing activation of the contractile proteins. While the events leading to initiation of Ca release have been well established and are known to involve activation of the SR Ca release channels/ryanodine receptors (RyR2s) by Ca that enters the cell through the sarcolemmal voltage-dependent Ca channels (i.e. Ca-induced Ca release, CICR), the mechanisms responsible for Ca release termination only begin to emerge. These restraining mechanisms are of particular importance in view of the propensity of CICR to spontaneous oscillations, a behavior known to cause cardiac arrhythmia. Growing evidence obtained recently in our laboratory indicates that restraining of CICR involves Ca signaling processes inside the SR lumen such that the decline of intra-SR Ca during release accounts for inactivation of the Ca release channels and channel refractoriness following their opening. From the luminal side of the SR membrane, RyR2s are complexed with a number of proteins including the Ca binding protein calsequestrin (CASQ2), and the putative anchoring proteins triadin and junctin. Together, these proteins form a macromolecular Ca signaling complex in the junctional SR. Recently, mutations in one of these proteins (CASQ2) have been linked to arrhythmia and sudden cardiac death inducible by exercise and emotional stress (catecholaminergic polymorphic ventricular tachycardia, CPVT). However, the role of these luminal auxiliary proteins in heart physiology and how their genetic defects lead to abnormal heart function is currently unknown. In the proposed studies we intend to: (1) define the modes of regulation of SR Ca release by the luminal auxiliary proteins CASQ2, triadin 1 and junctin, and (2) determine the molecular and cellular mechanisms of CPVT linked to mutations in CASQ2. We will use two complimentary state-of-the-art methodological approaches to accomplish these goals. The first approach involves a combination of adenoviral gene transfer strategies and methods of cellular physiology to analyze the effects of acute genetic manipulations of the luminal auxiliary proteins upon Ca handling and electrical activity in adult rat (rabbit) ventricular myocytes. The second approach employs an in vitro (i.e. lipid bilayer) system for functional reconstruction of the junctional complex from its individual components to further explore the individual functions of the luminal auxiliary proteins and their interactions among themselves and with the RyR2. The results obtained in the course of these studies will improve our understanding of the molecular basis of beat-to-beat regulation of Ca release in cardiac muscle and how molecular defects in proteins comprising the Ca release machinery lead to arrhythmia and sudden cardiac death. [unreadable] [unreadable]